Nucleic acid hybridization assays of various types are known. There are several assays that utilize a pair of DNA probes and a step of ligating the probes with a DNA ligase, wherein ligation requires that the probes be hybridized adjacent to one another on a target. In this application we use the term "binary probes assay" to refer generally to any assay that includes the step of ligating a pair of probes that are hybridized to a nucleic acid target adjacent to one another. The requirement that the pair of probes be hybridized only when adjacent to one another on a target means that ligation is "target-dependent." We refer to the pair of probes as "binary reporter probes" or "binary probes."
One binary probe assay is the ligase chain reaction ("LCR") (Barany, 1991). In LCR, a first pair of DNA binary probes is hybridized to one complementary strand of a DNA target and ligated there by a DNA-directed DNA ligase to form a first ligated product, and a second pair of DNA binary probes is hybridized to the first ligated product and similarly ligated to form a second ligated product. By cycling the reaction temperature, steps of melting, annealing probes and ligation are repeated to produce exponentially amplified product, i.e., ligated probes, that are then detected. We sometimes refer to ligated probes as a "reporter molecule" to distinguish from probes per se.
Another binary probe assay for DNA targets utilizes a pair of DNA binary probes, one of which serves to immobilize the target on the surface of a solid and the other of which contains a radioactive atom or fluorescent moiety (Landegren et al., 1988). This assay is reported to apply to RNA targets (using DNA binary probes), but no examples are given (Landegren & Hood, 1991).
A third assay for DNA targets utilizes a pair of DNA binary probes, one of which serves to immobilize the target on the surface of a solid, wherein the reporter molecule is a template that permits exponential amplification by an RNA-directed RNA polymerase such as bacteriophage Q.beta. replicase (Martinelli et al., 1992). The reporter molecule may be a DNA molecule that is itself a template for Q.beta. replicase (direct amplification), or it may be template for transcription by T7 RNA polymerase to produce an RNA template for Q.beta. replicase (indirect amplification).
The assays described above suffer from several drawbacks pertinent here. Most are for DNA targets, for example. That is a drawback, because RNA targets suitable for detection are in most cases much more abundant in samples than their corresponding DNA targets. All of the above assays use DNA binary probes. LCR requires thermal cycling and a thermocycler for amplification, and requires product analysis such as gel electrophoresis. The DNA binary probe assays of Landegren et al., 1988 and Landegren & Hood, 1991 do not include amplification and, therefore, are not sensitive assays. In assays employing exponential amplification by Q.beta. replicase (Martinelli et al, 1992) the use of DNA probes limits sensitivity: either an additional step of transcription is required, which increases cost, takes additional time, and lowers sensitivity, or a DNA reporter molecule must be amplified directly, which is very inefficient, thereby lowering sensitivity.
It is an objective of this invention to overcome the limitation of existing binary probe assays that use DNA binary probes.
It is a further objective of this invention to enable sensitive binary-probe assays for RNA targets using RNA probes and a stringent RNA-directed RNA ligase.